1. HIV-1 Antiretroviral Drug Resistance and Resistance Testing HIV and ARV resistance October 2009 Dr Michelle GordonOctober 2009

2. Evolution of Viral Mutations  Mutations arise because HIV-1 RT makes spontaneous errors (1 in 10 4 )  HIV-1 genome is 10 000 (10 4 ) bases long, therefore 1 error each time the genome is replicated  Production of virus = 10 9 to 10 10 virions per day  quasispecies  Every possible mutation present in quasispecies before ARV therapy Dr Michelle GordonOctober 2009

3. How drug resistance arises. Richman, DD. Scientific American, July 1998 How drug resistance arises Dr Michelle GordonOctober 2009

4. Resistant mutants emerge as a result of Resistant mutants emerge as a result of  genetic barrier (selective pressure of a regimen).  residual replication (potency of a regimen). Drugs differ with respect to their genetic barrier and time it takes to develop resistance. Drugs differ with respect to their genetic barrier and time it takes to develop resistance. 1 nt change → aa change → resistance = LOW genetic barrier 1 nt change → aa change → resistance = LOW genetic barrier >1 nt change at 1 st and 2nd codon position → aa change → resistance = LOW genetic barrier, but slightly longer to emerge >1 nt change at 1 st and 2nd codon position → aa change → resistance = LOW genetic barrier, but slightly longer to emerge >1 mutation (accumulation) → resistance = higher genetic barrier >1 mutation (accumulation) → resistance = higher genetic barrier Escape mutants will continue to replicate and develop additional (secondary/compensatory) mutations to: Escape mutants will continue to replicate and develop additional (secondary/compensatory) mutations to: further increase resistance (decreased drug susceptibility) further increase resistance (decreased drug susceptibility) increase viral fitness (“compensatory mutations”) increase viral fitness (“compensatory mutations”) Emergence of resistance Dr Michelle GordonOctober 2009

5. Disappearance of resistance  When treatment interrupted  No drug selection of quasispecies  Wild-type becomes dominant  Drug-resistant strains no longer detected by genotyping assays  Still minority population that can re- emerge if selection pressure re-applied Dr Michelle GordonOctober 2009

6. Antiretroviral drugs  5 classes drugs  PR Inhibitors (PIs) (9)  Nucleoside/nucleotide RT Inhibitors (NRTIs) (8)  Non-nucleoside RT Inhibitors (NNRTIs) (4)  Fusion Inhibitors (2)  Integrase Inhibitors (1)  Current therapies imperfect because:  Regimen complexities  Toxicities  Drug resistance Dr Michelle GordonOctober 2009

7. Relationship between drug concentration, viral suppression and adverse effects  adequate drug concentrations are need in order to bring about desired pharmacological and virological effects. Dr Michelle GordonOctober 2009

8. Targets HIV-1 PRTargets HIV-1 PR Attaches to the PR binding cleft that recognizes and cleaves precursor polyproteins.Attaches to the PR binding cleft that recognizes and cleaves precursor polyproteins.  Most PR resistance mutations alter the structure of the substrate cleft  Causes resistance by reducing the binding affinity between the inhibitor and the mutant protease enzyme.  Mutations also occur in the flaps compensate for loss of fitness compensate for loss of fitness PIs and Resistance Dr Michelle GordonAugust 2006

9. PI drug interactions  PIs are metabolized by CYP 3A4 and P450 (the body’s drug clearance mechanisms).  Ritonovir (RTV) is one of the most potent CYP 3A4 inhibitors known.  Most PIs are co-administered with sub-therapeutic doses of RTV thereby increasing the plasma levels of the PI.  Boosted PI levels can overcome small reductions in susceptibility conferred by early PI mutations, thus prolonging viral suppression.  NNRTI’s (NVP and EFV) and TB drugs are inducers of CYP 450 and affect PI concentrations. Dr Michelle GordonOctober 2009

10. NRTIs NRTIs are ddNTPs NRTIs are ddNTPs  Phosphorylated NRTIs compete with natural dNTPs for incorporation into the newly synthesized DNA chains - cause chain termination Nucleosides require 3 phosphates; nucleotides require 2 phosphates Nucleosides require 3 phosphates; nucleotides require 2 phosphates Dr Michelle GordonOctober 2009

11. Chain termination with NRTIs If AZT was the last nt added, then the RT enzyme cannot add new nucleotides Dr Michelle GordonOctober 2009

12. Pyrophosphorylation After incorporation of a nucleoside, two released phosphates may attack the link, causing the nucloside to be released again. After incorporation of a nucleoside, two released phosphates may attack the link, causing the nucloside to be released again. NRTIs and Resistance Dr Michelle GordonOctober 2009

13.  The non-nucleoside RT inhibitors (NNRTIs) bind to a hydrophobic pocket (called the NNRTI-binding hydrophobic pocket (called the NNRTI-binding pocket) pocket)  NNRTIs inhibit replication by displacing the enzyme active site relative to the polymerase binding site  A single mutation in the NNRTI-binding pocket may result in high-level resistance to one or more may result in high-level resistance to one or more NNRTIs (low genetic barrier). NNRTIs (low genetic barrier). NNRTIs and Resistance Dr Michelle GordonOctober 2009

14. Fusion Inhibitors During HIV-1 infection, the virus’s gp120 binds to both a CD4 and chemokine receptor (CCR5 or CXCR4) on the target cells During HIV-1 infection, the virus’s gp120 binds to both a CD4 and chemokine receptor (CCR5 or CXCR4) on the target cells causes a conformational change in gp120 causes a conformational change in gp120 promotes the fusion of the viral and cellular membranes promotes the fusion of the viral and cellular membranes Dr Michelle GordonOctober 2009

15. This class of drugs interferes with the binding, fusion and entry of an HIV virion to a human (host) cell. Enfuvirtide (Fuseon) Binds to gp41 transmembrane protein and blocks fusion of hiv-1 to the host cell. Binds to gp41 transmembrane protein and blocks fusion of hiv-1 to the host cell. Maraviroc (Selzentry; Celsentri ) Binds to CCR5, preventing an interaction with gp120. Binds to CCR5, preventing an interaction with gp120. Fusion Inhibitors Dr Michelle GordonOctober 2009

16. Integrase Inhibitors Integrase is one of three viral enzymes necessary for HIV replication that integrates or blends HIV genetic material into the DNA of human CD4 cells. Integrase is one of three viral enzymes necessary for HIV replication that integrates or blends HIV genetic material into the DNA of human CD4 cells. This makes it possible for the infected cell to make new copies of HIV This makes it possible for the infected cell to make new copies of HIV Isentress (raltegravir) By interfering with integrase, the integrase inhibitors prevent HIV genetic material from integrating into the CD4 cell, thus stopping HIV replication. By interfering with integrase, the integrase inhibitors prevent HIV genetic material from integrating into the CD4 cell, thus stopping HIV replication. Dr Michelle GordonOctober 2009

17. Mutations associated with resistance to PIs Dr Michelle GordonOctober 2009 http://hivdb.stanford.edu/

18. Mutations associated with resistance toNRTIs Mutations associated with resistance to NRTIs Dr Michelle GordonOctober 2009 http://hivdb.stanford.edu/

19. Mutations associated with resistance to NNRTIs Dr Michelle GordonOctober 2009 http://hivdb.stanford.edu/

20. Mutations associated with resistance to Fusion Inhibitors Dr Michelle GordonOctober 2009 http://hivdb.stanford.edu/

21. Mutations associated with resistance to Integrase Inhibitors Dr Michelle GordonOctober 2009 http://hivdb.stanford.edu/

22. How do you measure drug resistance? Genotyping: Indirect assay: Detects drug resistance mutations that are present in the relevant virus genes. Phenotyping: Direct assay: Measures the ability of the virus to grow in various concentrations of antiretroviral drugs. Dr Michelle GordonOctober 2009

23. Resistance Testing 1. Genotypic Testing More commonly used in clinical settings wider availability, lower cost and quicker turnaround (1-2 weeks) PI and RTIS PI and RTIS HIV-1 GenotypR PLUS (Specialty Laboratories) HIV-1 GenotypR PLUS (Specialty Laboratories) TRUGENE HIV-1 genotyping test (Bayer) TRUGENE HIV-1 genotyping test (Bayer) VircoGEN II (Virco) VircoGEN II (Virco) Viroseq HIV genotyping system (Abbott Diagnostics) Viroseq HIV genotyping system (Abbott Diagnostics) GeneSeq (ViroLogic) GeneSeq (ViroLogic) HIV-1 Mutation Analysis (Focus Technologies) HIV-1 Mutation Analysis (Focus Technologies) HIV ViroTYPE (Rheumatology Diagnostoc Laboratory) HIV ViroTYPE (Rheumatology Diagnostoc Laboratory) GenoSure (LabCorp and Virco) GenoSure (LabCorp and Virco) Fusion Inhibitors Fusion Inhibitors TRUGENE HIV-1 Envelope (gp41) Genotyping Assay (Bayer) TRUGENE HIV-1 Envelope (gp41) Genotyping Assay (Bayer) HIV GenoSure Fusion (LabCorp) HIV GenoSure Fusion (LabCorp) Dr Michelle GordonOctober 2009

24. Genotyping: Advantages Identification of all nucleotides, amino acid differences, deletions & insertions Genotyping has the ability to detect resistant virus that constitutes only a small proportion (about 20%) of the viral population. This can provide “predictive” early warning of developing resistance before full resistance develops Faster and less expensive than phenotype assay Dr Michelle GordonOctober 2009

25. Genotyping: Disadvantages Reports may be difficult to interpret unless clinician is very experienced Labs use different software programs to predict resistance - need a consensus on which mutations are important There is a lot of variation in the quality of the “product” from different laboratories especially in the ability to detect minority species in the population Current limitation of use is that viral load needs to  1000 copies/ml - need greater sensitivity Dr Michelle GordonOctober 2009

26. Phenotyping: Advantages Provides resistance information on each drug regardless of the presence of multiple mutations Interpretation may be more intuitive than for genotype assay Very useful in patients with complex drug history and complicated mutation profile Very useful for deciphering cross-resistance May be more useful than genotyping for new drugs until appropriate mutations are established by clinical data Dr Michelle GordonOctober 2009

27. Phenotyping: Disadvantages If drug resistant population is minor the phenotypic effect may not be detected Current limitation of use is that viral load needs to  1000 copies/ml - need greater sensitivity Very expensive and time-consuming The relevance of small changes in drug sensitivity not yet fully determined - drugs to which patient is actually still sensitive may be unnecessarily eliminated Dr Michelle GordonOctober 2009

28. There is continued effort to develop cheaper in-house assays for resistance genotyping. However, there needs to be some mechanism of standardization between laboratories. Also, because all in-house methods are nested reactions (with an increased chance of contamination) there needs to be the same level of adherence to quality control. In-house genotyping assays

29. Genotyping kits Already optimized Already optimized Standardized Standardized Built-in QC procedures Built-in QC procedures Thus, prevent possible sample to sample contamination Thus, prevent possible sample to sample contamination Very expensive Very expensive Insensitive to presence of minor variants Insensitive to presence of minor variants Interpretation requires prior knowledge of genetic determinants of resistance Interpretation requires prior knowledge of genetic determinants of resistance In-house assays Most require optimization Most require optimization Not standardized btw labs Not standardized btw labs Not usually have built-in QC Not usually have built-in QC Increased risk of contamination Increased risk of contamination Much cheaper Much cheaper More sensitive, because a nested reaction More sensitive, because a nested reaction Interpretation the same Interpretation the same Kits vs In-house assays

30. 1.8kb Load samples onto ABI 3100 A B C F G H protease RT plasma dried blood spot Genotyping using the Viroseq Kit Dr Michelle GordonOctober 2009

31. Sequence Analysis Dr Michelle GordonOctober 2009

32. Interpretation of Genotypic Resistance Tests Interpretation complicated by complex patterns of mutationsInterpretation complicated by complex patterns of mutations 2 basic approaches: i)Rules-based algorithms: Developed by experts; Based on published data on phenotypic impact and clinical significance of drug resistance mutations TruGene (Bayer)TruGene (Bayer) Viroseq (Celera)Viroseq (Celera) HIVdb (Stanford)HIVdb (Stanford) ANRS (French)ANRS (French) Rega (Belgium)Rega (Belgium) Dr Michelle GordonOctober 2009

33. ii) Machine-learning algorithms Contain rules discovered by a computer program by analyzing data linking genotype to phenotype or clinical outcome. Learn from a training data set and test performance using a test data set Decision treesDecision trees Categorical analysis and regression trees (CART)Categorical analysis and regression trees (CART) Neural networksNeural networks Virtual phenotypeVirtual phenotype Interpretation of Genotypic Resistance Tests Dr Michelle GordonOctober 2009

34. http://hivdb.stanford.edu/

40. Limitations of Drug Resistance Testing i) Relationship between drug resistance and clinical failure is complex Non-adherence Non-adherence Sub-optimal regimens Sub-optimal regimens Pharmacokinetics Pharmacokinetics Host factors Host factors Many drug resistant variants are less fit Many drug resistant variants are less fit ii) Complex quasispecies iii) Cross-resistance iv) Potentially miss minor populations Dr Michelle GordonOctober 2009

41.  Adherance  New Drugs  Salvage Therapy  Intensification Overcoming Resistance Dr Michelle GordonOctober 2009

42. Conclusion Resistance-conferring mutations occur continuously, in absence and presence of drug therapy. Resistance-conferring mutations occur continuously, in absence and presence of drug therapy. Occur more rapidly when viral load is high: more replication, more mutations. Occur more rapidly when viral load is high: more replication, more mutations. Mutant strains become dominant under drug selection pressure. Mutant strains become dominant under drug selection pressure. Resistance testing gives information on which drugs no longer potent in regimen. Resistance testing gives information on which drugs no longer potent in regimen. Resistance testing limited because relationship between drug resistance and clinical failure is complex; may miss minor populations; cross-resistance. Resistance testing limited because relationship between drug resistance and clinical failure is complex; may miss minor populations; cross-resistance. Understanding resistance is important for optimal patient management! Understanding resistance is important for optimal patient management! Dr Michelle GordonOctober 2009

43. References http://hivdb.stanford.edu http://www.hivresistanceweb.com http://hiv.medscape.com/Home/Topics/AIDS/AIDS.html http://www.hivatis.org http://hiv-lanl.gov/seq-db.html Dr Michelle GordonOctober 2009